Competitive and Synergistic Interactions of G Protein β2 and Ca2+ Channel β1b Subunits with Cav2.1 Channels, Revealed by Mammalian Two-hybrid and Fluorescence Resonance Energy Transfer Measurements
2003; Elsevier BV; Volume: 278; Issue: 49 Linguagem: Inglês
10.1074/jbc.m306645200
ISSN1083-351X
AutoresAlexander Hümmer, Oliver Delzeith, Shannon R. Gomez, Rosa L. Moreno, Melanie D. Mark, Stefan Herlitze,
Tópico(s)Receptor Mechanisms and Signaling
ResumoPresynaptic Ca2+ channels are inhibited by metabotropic receptors. A possible mechanism for this inhibition is that G protein βγ subunits modulate the binding of the Ca2+ channel β subunit on the Ca2+ channel complex and induce a conformational state from which channel opening is more reluctant. To test this hypothesis, we analyzed the binding of Ca2+ channel β and G protein β subunits on the two separate binding sites, i.e. the loopI–II and the C terminus, and on the full-length P/Q-type α12.1 subunit by using a modified mammalian two-hybrid system and fluorescence resonance energy transfer (FRET) measurements. Analysis of the interactions on the isolated bindings sites revealed that the Ca2+ channel β1b subunit induces a strong fluorescent signal when interacting with the loopI–II but not with the C terminus. In contrast, the G protein β subunit induces FRET signals on both the C terminus and loopI–II. Analysis of the interactions on the full-length channel indicates that Ca2+ channel β1b and G protein β subunits bind to the α1 subunit at the same time. Coexpression of the G protein increases the FRET signal between α1/β1b FRET pairs but not for α1/β1b FRET pairs where the C terminus was deleted from the α1 subunit. The results suggest that the G protein alters the orientation and/or association between the Ca2+ channel β and α12.1 subunits, which involves the C terminus of the α1 subunit and may corresponds to a new conformational state of the channel. Presynaptic Ca2+ channels are inhibited by metabotropic receptors. A possible mechanism for this inhibition is that G protein βγ subunits modulate the binding of the Ca2+ channel β subunit on the Ca2+ channel complex and induce a conformational state from which channel opening is more reluctant. To test this hypothesis, we analyzed the binding of Ca2+ channel β and G protein β subunits on the two separate binding sites, i.e. the loopI–II and the C terminus, and on the full-length P/Q-type α12.1 subunit by using a modified mammalian two-hybrid system and fluorescence resonance energy transfer (FRET) measurements. Analysis of the interactions on the isolated bindings sites revealed that the Ca2+ channel β1b subunit induces a strong fluorescent signal when interacting with the loopI–II but not with the C terminus. In contrast, the G protein β subunit induces FRET signals on both the C terminus and loopI–II. Analysis of the interactions on the full-length channel indicates that Ca2+ channel β1b and G protein β subunits bind to the α1 subunit at the same time. Coexpression of the G protein increases the FRET signal between α1/β1b FRET pairs but not for α1/β1b FRET pairs where the C terminus was deleted from the α1 subunit. The results suggest that the G protein alters the orientation and/or association between the Ca2+ channel β and α12.1 subunits, which involves the C terminus of the α1 subunit and may corresponds to a new conformational state of the channel. Voltage-gated Ca2+ channels of the N-, P/Q-, and R-type are inhibited by G protein-coupled receptors. The voltage-dependent inhibition is mediated by G protein βγ subunits (1.Herlitze S. Garcia D.E. Mackie K. Hille B. Scheuer T. Catterall W.A. Nature. 1996; 380: 258-262Crossref PubMed Scopus (706) Google Scholar, 2.Ikeda S.R. Nature. 1996; 380: 255-258Crossref PubMed Scopus (710) Google Scholar) but is also induced by coexpression of the G protein β subunit alone (1.Herlitze S. Garcia D.E. Mackie K. Hille B. Scheuer T. Catterall W.A. Nature. 1996; 380: 258-262Crossref PubMed Scopus (706) Google Scholar). Ca2+ channels consist of at least three subunits: the pore-forming α1 and several auxiliary subunits such as the intracellulary located β subunit and the transmembrane subunit α2δ. The α1 subunit consists of four channel domains, which are connected via intracellular peptide loops (for review see Refs. 3.Catterall W.A. Annu. Rev. Cell Dev. Biol. 2000; 16: 521-555Crossref PubMed Scopus (1958) Google Scholar, 4.Dolphin A.C. Page K.M. Berrow N.S. Stephens G.J. Canti C. Ann. N. Y. Acad. Sci. 1999; 868: 160-174Crossref PubMed Scopus (8) Google Scholar, 5.Jarvis S.E. Zamponi G.W. Trends Pharmacol. Sci. 2001; 22: 519-525Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). G protein βγ subunits modulate the channel via interaction with the intracellular peptide domain (loop I–II) and the C terminus of the α1 subunit (6.Zhang J.F. Ellinor P.T. Aldrich R.W. Tsien R.W. Neuron. 1996; 17: 991-1003Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 7.De Waard M. Liu H. Walker D. Scott V.E. Gurnett C.A. Campbell K.P. Nature. 1997; 385: 446-450Crossref PubMed Scopus (374) Google Scholar, 8.Herlitze S. Hockerman G.H. Scheuer T. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1512-1516Crossref PubMed Scopus (169) Google Scholar, 9.Qin N. Platano D. Olcese R. Stefani E. Birnbaumer L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8866-8871Crossref PubMed Scopus (207) Google Scholar, 10.Zamponi G.W. Bourinet E. Nelson D. Nargeot J. Snutch T.P. Nature. 1997; 385: 442-446Crossref PubMed Scopus (406) Google Scholar, 11.Page K.M. Stephens G.J. Berrow N.S. Dolphin A.C. J. Neurosci. 1997; 17: 1330-1338Crossref PubMed Google Scholar, 12.Furukawa T. Miura R. Mori Y. Strobeck M. Suzuki K. Ogihara Y. Asano T. Morishita R. Hashii M. Higashida H. Yoshii M. Nukada T. J. Biol. Chem. 1998; 273: 17595-17603Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Interestingly, the Gβγ subunit-binding sites overlap with the Ca2+ channel β subunit-binding sites on the α1 channel subunit (5.Jarvis S.E. Zamponi G.W. Trends Pharmacol. Sci. 2001; 22: 519-525Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 7.De Waard M. Liu H. Walker D. Scott V.E. Gurnett C.A. Campbell K.P. Nature. 1997; 385: 446-450Crossref PubMed Scopus (374) Google Scholar, 9.Qin N. Platano D. Olcese R. Stefani E. Birnbaumer L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8866-8871Crossref PubMed Scopus (207) Google Scholar, 10.Zamponi G.W. Bourinet E. Nelson D. Nargeot J. Snutch T.P. Nature. 1997; 385: 442-446Crossref PubMed Scopus (406) Google Scholar). In addition to the overlapping binding sites, G protein βγ and Ca2+ channel β subunits induce antagonistic effects on defined biophysical properties of the channel. For example, Ca2+ channel β subunits (with the exception of β2 subunits) shift the voltage dependence of activation to more hyperpolarized potentials, whereas Gβγ subunits have the opposite effects, i.e. a depolarizing shift (e.g. Refs. 13.Bourinet E. Soong T.W. Stea A. Snutch T.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1486-1491Crossref PubMed Scopus (219) Google Scholar, 14.Dolphin A.C. J. Physiol. (Lond.). 1998; 506: 3-11Crossref Scopus (233) Google Scholar, 15.Hille B. Trends Neurosci. 1994; 17: 531-536Abstract Full Text PDF PubMed Scopus (672) Google Scholar, 16.Ikeda S.R. Dunlap K. Adv. Second Messenger Phosphoprotein Res. 1999; 33: 131-151Crossref PubMed Google Scholar, 17.Jones S.W. Elmslie K.S. J. Membr. Biol. 1997; 155: 1-10Crossref PubMed Scopus (53) Google Scholar, 18.Birnbaumer L. Qin N. Olcese R. Tareilus E. Platano D. Costantin J. Stefani E. Bioenerg. Biomembr. 1998; 30: 357-375Crossref PubMed Scopus (202) Google Scholar). The overlapping binding sites on the channel as well as their antagonistic effects on the voltage dependence of channel activation suggest that Ca2+ channel β and G protein βγ subunits may compete for binding sites on the α1 subunits during G protein modulation. Early biophysical analysis of the Ca2+ channel G protein modulation suggested that Ca2+ channels are stabilized in a certain conformational state during G protein modulation from which channel opening is more difficult to achieve (19.Bean B.P. Nature. 1989; 340: 153-156Crossref PubMed Scopus (672) Google Scholar). According to this model, G protein βγ subunits may induce and stabilize this reluctant state of the channel (20.Elmslie K.S. Zhou W. Jones S.W. Neuron. 1990; 5: 75-80Abstract Full Text PDF PubMed Scopus (289) Google Scholar, 21.Boland L.M. Bean B.P. J. Neurosci. 1993; 13: 516-533Crossref PubMed Google Scholar, 22.Patil P.G. de Leon M. Reed R.R. Dubel S.J. Snutch T.P. Yue D.T. Biophys. 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Green fluorescent protein (GFP) 1The abbreviations used are: GFPgreen fluorescent proteinaaamino acid(s)ADactivation domainAIDα1 interaction domainBDbinding domainCFPcyan fluorescent proteinFRETfluorescence resonance energy transferHEKhuman embryonic kidneyMLRmultilinear regressionMTHmammalian two-hybrid systemNLSnuclear localization signalYFPyellow fluorescent proteinOKopossum kidneyGTPγSguanosine 5′-3-O-(thio)triphosphate. has become an important fluorescent tag to study the localization, targeting, and interaction of proteins (26.Tsien R.Y. Annu. Rev. Biochem. 1998; 67: 509-544Crossref PubMed Scopus (4983) Google Scholar). Visualization of GFP does not require any cofactor or enzymatic reaction and is therefore suitable as a reporter gene for an immediate read out in a two-hybrid interaction assay, for example. Because of the existence of several spectrally distinguishable variants of GFP, two reporter genes can be used to record and compare the expression and interaction of two independent proteins. Dual-color imaging and fluorescence resonance energy transfers (FRET) were performed in various studies with promising results by using CFP and YFP as fluorescent pairs (27.Miyawaki A. Llopis J. Heim R. McCaffery J.M. Adams J.A. Ikura M. Tsien R.Y. Nature. 1997; 388: 882-887Crossref PubMed Scopus (2650) Google Scholar, 28.Ellenberg J. Lippincott-Schwartz J. Presley J.F. Trends Cell Biol. 1999; 9: 52-56Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 29.Truong K. Ikura M. Curr. Opin. Struct. Biol. 2001; 11: 573-578Crossref PubMed Scopus (289) Google Scholar, 30.Ruiz-Velasco V. Ikeda S.R. J. Physiol. (Lond.). 2001; 537: 679-692Crossref Scopus (39) Google Scholar). green fluorescent protein amino acid(s) activation domain α1 interaction domain binding domain cyan fluorescent protein fluorescence resonance energy transfer human embryonic kidney multilinear regression mammalian two-hybrid system nuclear localization signal yellow fluorescent protein opossum kidney guanosine 5′-3-O-(thio)triphosphate. By using a modified mammalian two-hybrid system (MTH) and FRET, we asked how Ca2+ channel β and G protein β subunits interact at the binding sites on the α1 subunit and whether the G protein induces a new conformational state of the channel. Our results suggest that Ca2+ channel β1b and G protein β subunits differentially interact with the two isolated binding sites of the α1 subunit (i.e. loopI–II and C terminus). On the functional full-length channel the Ca2+ channel β1b subunits may interact more strongly than G protein β subunits, because the FRET signals were larger for α1/β1b FRET pairs. Interestingly, when G protein β subunits were coexpressed with Ca2+ channel α1 and β subunits, there was an increase in the fluorescence signal between the Ca2+ channel subunits. This increase in FRET was abolished when the C terminus was deleted from the α1 subunit or overexpressed in the untagged form. The results suggest that the G protein induces an altered conformational state of the P/Q-type channel, which probably involves the binding of the G protein to the C terminus of the α1 subunit. Construction of DNA-BD and AD Fusion Proteins and pHASH-3—The following cloning and reporter vectors of the mammalian Matchmaker two-hybrid assay kit (Clontech) were used: pM as cloning vector to construct the GAL4 DNA-BD fusion constructs; pVP16 as cloning vector for AD fusion constructs; pM3-VP16, pM-53, and pVP16-T as positive control vectors; pVP16 and pM without insert as negative control vectors. Gβ2, Gγ3, and the Ca2+ channel constructs β1b, α12.1-loop I–II, and α11.2-loop I–II were amplified by a single PCR, and constructs were subcloned in-frame into pM and pVP16. All amplified products were verified by sequencing. For construction of pHASH-3, five consensus GAL4-binding sites (UASG17-mer (x5)) and an adenovirus E1b minimal promoter were amplified by a single PCR and were subcloned into pEYFP-C1 (Clontech). This vector was called pHASH-1. Then two nuclear localization signals were added 3′ in-frame into the YFP gene. This vector was called pHASH-2. For creating pHASH-3, two nuclear localization signals were also added 3′ to the CFP gene in pECFP (pECFP-NLS). The inducible YFP gene was cut out from pHASH2 using BspTI and subsequently subcloned into the BspTI site of pECFP-NLS. The 5′ BspTI site was introduced into pHASH-2 with the inducible promoter via PCR. Cell Culture and Immunohistochemistry—Opossum kidney (OK), Chinese hamster ovary, and human embryonic kidney (HEK) 293 cells were transfected with the indicated DNAs in each set of experiments with Effectene™ Transfection Reagent (Qiagen GmbH, Germany). 0.25 μg of each DNA in combination with 0.5 μg of the reporter plasmids (pHASH-3) were used for transfection. Equal amounts of total DNA within one set of experiments were used by adding unrelated DNA (plasmid pBF-1) to the transfection mixture when necessary. For example in Fig. 1 a maximum of 5 different DNAs were transfected in a 2:1:1:1:1 (with pHASH-3 added at twice the molar ratio than the other DNAs). When only 3 or 4 different DNAs were transfected, the missing DNA was replaced by pBF-1 DNA at the same ratio. Western blot analysis of transfected OK cells were performed with a polyclonal anti-Gal4 binding domain antibody and a monoclonal anti-VP-16 activation domain antibody (Santa Cruz Biotechnology, Santa Cruz, CA) according to standard procedures as described by Mark et al. (31.Mark M.D. Liu Y. Wong S.T. Hinds T.R. Storm D.R. J. Cell Biol. 1995; 130: 701-710Crossref PubMed Scopus (64) Google Scholar). Immunocytochemistry and Quantification of YFP and CFP Signals—Cells were embedded in Fluoromount (133 mm Tris/HCl, 30% glycerol; 11% Mowiol, 2% diazabicyclo[2.2.2]octane). Fluorescence was detected with a conventional fluorescence microscope (Axioskop; Carl Zeiss, Oberkochen, Germany). For CFP and YFP detection, the following filter sets were used: CFP, excitation, short-pass D436/10; beamsplitter 460DCLP and emission, bandpass filter 480/30; YFP, excitation, short-pass HQ 500/20; beamsplitter Q515LP and emission, bandpass filter 535/30. All filters were obtained from AHF Analysentechnik AG, Germany. Intensity ratios between nuclear YFP and CFP fluorescence were calculated by dividing the mean intensity values for YFP by the mean intensity values for CFP. Mean intensity values of YFP and CFP fluorescence were calculated by subtracting the intensity values measured from the extracellular background from the intensity values measured from the fluorescence in the nucleus of the individual cells. Intensity values are defined as the sum of the gray scale values for all pixels contained in a defined object area. For every fluorophore in each set of experiments the optimal exposure time for the YFP and the CFP fluorescence signals was determined for the strongest signal of the positive control (i.e. pM53/pVP16-T/pHASH-1–3). Fluorescence intensities were compared with the fluorescence signal at the defined exposition time. In addition, CFP fluorescence background after YFP excitation was measured by expressing pHASH-3 alone, and values were subtracted from all experiments performed for the same transfection. Images were captured with a CCD camera (RTE/CCD-1300-Y/HS Princeton Instruments; Tucson, AZ), and pictures were analyzed with MetaMorph 4.01 (Visitron Systems GmbH, Puchheim, Germany). All experiments described were performed at least in triplicate, and data were presented as means ± S.E. Colors used for YFP, CFP, and DAPI in Fig. 1 are computer-generated colors (Adobe Photoshop 5.5). Constructs—Constructs α12.1-loopI–II (residues 369–418), α12.1-loopI–II-Y/S, α11.2-loopI–II (residues 406–520), α12.1-C terminus (residues 1766–2212), α12.1 full-length, α12.1 full-length-ΔC terminus (residues 1–1857), β1b, β4, Gβ2, Gγ3 were either PCR-amplified or if restriction sites were suitable cloned into either pECFP-C1, pEYFP-C1 (Clontech), pECFP-C2, and pEYFP-C2 (derived from pEGFP-C2) or as non-tagged versions into pcDNA1, -3, or pcDNA3.1. All PCR-amplified products were verified by sequencing. FRET Measurements—For the calculation of FRET values and FRET-derived values, a two-step approach was used, which is based on the formalism and procedures of Erickson et al. (32.Erickson M.G. Alseikhan B.A. Peterson B.Z. Yue D.T. Neuron. 2001; 31: 973-985Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). In the first step, the constants RD1 and RA1 were determined by a multilinear regression (MLR) of the type FRETfl = α × CFPfl + β × YFPfl + γ [MLR] (where fl indicates fluorescence). A simple manipulation of (FRET = (FRETfl - RD1 × CFPfl)/(RA1 × (YFPfl - RD2 × CFPfl))) yields the relations RD1 = α + FRET × RA1 × RD2 and RA1 = β/FRET. Because the term FRET × RA1 × RD2 turns out to be exceedingly small in comparison to RD1 (see Table in the Supplemental Material), the constant RD1 was estimated by α to a good degree of approximation. This regression method has the advantage of producing the results completely independent of any additive adjustments of the basic input data usually necessary because of background variation. Furthermore, the correlation coefficient r of [MLR] can be calculated. In case r is close to 1or -1, it indicates the appropriateness of a linear relation between the variables, as predicted by the theory given in Erickson et al. (32.Erickson M.G. Alseikhan B.A. Peterson B.Z. Yue D.T. Neuron. 2001; 31: 973-985Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). Considering the data of cells expressing donor (X-CFP) only, the constant RD1 was set to be α in [MLR]. By using the data of the cells expressing acceptor protein (X-YFP) only, RA1 was determined as β of the [MLR], because in this case the FRET ratio (FRET) is equal to 1 by definition (FRET = (FRETfl (from FRET pair) + FRETfl (from YFP))/FRETfl (from YFP)) (32.Erickson M.G. Alseikhan B.A. Peterson B.Z. Yue D.T. Neuron. 2001; 31: 973-985Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). In the second step the constants ΔFRmax and FRETmax were calculated by an ordinary linear fit of the data of cells expressing both donor (X-CFP) and acceptor protein (X-YFP). As suggested by equation FRET = ΔFRmax × Ab + 1 (32.Erickson M.G. Alseikhan B.A. Peterson B.Z. Yue D.T. Neuron. 2001; 31: 973-985Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar), the data were linearly fitted according to the free type FRET = m × Ab + c (y = mx + q). The predicted value FRET of the FRET ratio was given according to FRET = (FRETfl - RD1 × CFPfl)/(RA1 × (YFPfl - RD2 × CFPfl)) (see above) (32.Erickson M.G. Alseikhan B.A. Peterson B.Z. Yue D.T. Neuron. 2001; 31: 973-985Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar) using the original data and the R constants calculated in the first step. The percentage Ab of bound acceptors was calculated according to Ab = (CFPest + YFPest + Kd(Eff) - ((CFPest + YFPest + Kd(EFF))2 - 4 × CFPest × YFPest)1/2]/(2 × YFPest) [A34] (with CFPest = CFPfl/M_D; YFPest = YFPfl/M_A; M_A and M_D set as in Erickson et al. (32.Erickson M.G. Alseikhan B.A. Peterson B.Z. Yue D.T. Neuron. 2001; 31: 973-985Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar), KD(EFF) set 0), where Eff indicates efficiency and est indicates estimate. The quality of the linear fit was measured by the correlation coefficient r shown in the figures. Throughout the experiments statistical significance (p) was determined with a two-tailed Student's t test with p < 0.05 (*) and p < 0.01 (**). Standard errors are the mean ± S.E. Electrophysiology—CFP- and YFP-tagged Ca2+ channel subunits (α12.1, β1b) and the G protein subunit (Gβ2) were coexpressed in tsA201 cells, and Ca2+ channel-mediated Ba2+ currents were measured and analyzed as described previously (1.Herlitze S. Garcia D.E. Mackie K. Hille B. Scheuer T. Catterall W.A. Nature. 1996; 380: 258-262Crossref PubMed Scopus (706) Google Scholar, 33.Mark M.D. Wittemann S. Herlitze S. J. Physiol. (Lond.). 2000; 528: 65-77Crossref Scopus (30) Google Scholar, 34.Wittemann S. Mark M.D. Rettig J. Herlitze S. J. Biol. Chem. 2000; 275: 37807-37814Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). A Modified Mammalian Two-hybrid System to Detect Protein-Protein Interactions—The yeast two-hybrid system is based on the finding by Fields and Song (35.Fields S. Song O. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4880) Google Scholar) that eukaryotic trans-acting transcription factors like GAL4 can be divided into two physically separated but still functional independent domains. Both domains are normally part of a nuclear protein, which binds to a specific activation sequence of the target genes and regulates their transcription. Therefore, the DNA binding domain (DNA-BD) binds to certain upstream activating sequences (UAS) in close proximity to the promoter of the gene. One or several activation domains (AD) increase the transcription rate by directing the RNA polymerase II complex for downstream action. The AD and DNA-BD have to be in close physical proximity for efficient gene transcription. Separating AD from DNA-BD results in loss of gene transcription, whereas tethering AD to the DNA-BD by fusion of interacting proteins restores the function of the transcription factor. The possibility to separate the two domains and fuse them to putative interacting proteins allows one to monitor the interaction via expression of a certain reporter gene (36.Fields S. Sternglanz R. Trends Genet. 1994; 10: 286-292Abstract Full Text PDF PubMed Scopus (526) Google Scholar) (Clontech and Stratagene). Recently, GFP has been used for monitoring protein interactions in an MTH system, which is based on the same principles as the yeast system (37.Shioda T. Andriole S. Yahata T. Isselbacher K.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5220-5224Crossref PubMed Scopus (26) Google Scholar). One problem concerning the detection of especially weak protein interactions in the MTH system is the identification of positively transfected cells and the detection of a signal relative to background. In addition, the induced fluorescence intensity among several cells within a single assay may vary because of unequal numbers of reporter plasmids within the cell, different expression times after transfection, or because of the cell type. To overcome these problems, we introduced a second constitutively expressed reporter, the cyan fluorescence protein (CFP), into a vector where YFP is under the control of the GAL4-inducible promoter. The CFP is under CMV promoter control and is also transported to the nucleus. CFP-mediated fluorescence therefore indicates a positively transfected cell. The induction of YFP fluorescence can now be compared with the fluorescence signals of CFP, which are both expressed in the same restricted area, i.e. the nucleus (Fig. 1a). Thus, induced YFP signals can be detected and monitored relative to the CFP signals within single cells. As shown in Fig. 1, b–d, we first determined the optimal expression time for detection of protein-protein interactions in the MTH system. This was necessary because the YFP reporter plasmid gene reveals low expression over time in the absence of interacting proteins. We therefore analyzed the signal ratio between YFP-induced fluorescence and the constitutive CFP fluorescence for positive and negative controls at various expression times after transfection. As shown in Fig. 1, b–d, the signal ratio between YFP and CFP fluorescence depends on the incubation time after transfection. YFP fluorescence was first detected 12 h after transfection in 20–50% of constitutively CFP-expressing cells for the positive control constructs, i.e. pM3-VP16 (fused GAL-4-DNA-BD and VP16-AD), interacting p53 protein/SV40 large T-antigen (pM53/pVP16-T), and Gβ2/Gγ3 interaction (Fig. 1, b–e). The relative cell number in which YFP fluorescence was detected in CFP-positive cells increased to 75–100% 24 h after transfection (Fig. 1b). In contrast, for the negative controls (non-interacting pairs pM/pVP16, pM53/pVP16, and pHASH-3 alone), YFP fluorescence was only detected after 18 h in 15% of the CFP-positive cells, and the cell number with YFP fluorescence further increased to a saturating level after 36–48 h of expression (Fig. 1c). Forty-eight h after transfection 60–70% of the CFP-positive cells revealed YFP fluorescence due to the leakage of the GAL4/E1b promoter (Fig. 1c). As shown in Fig. 1d, the optimal signal to noise ratio for the detection of YFP fluorescence and quantification of protein interactions occurred after 18–24 h of expression for OK cells. To demonstrate further that the signal to noise ratio decreased for incubation times longer than 24 h, we compared the YFP/CFP fluorescence ratios from cells incubated for 18 and 36 h after transfection. As shown in Fig. 1, e and f, the YFP/CFP fluorescence intensity ratios decline significantly for Gβ2/Gγ3 interaction from 0.52 ± 0.08 (n = 21) to 0.29 ± 0.05 (n = 54) (p < 0.05, Student's t test). Thus, optimal YFP/CFP fluorescence ratios are obtained between 18 and 24 h following transfection, which is the time where background fluorescence is minimized. Interaction of the Ca2+Channel β Subunit and the G Protein β Subunit with the Intracellular Loop I–II of the Ca2+Channel α1Subunits—The binding sites of the Ca2+ channel β and G protein βγ subunits are localized at the intracellular domain connecting domain I and II and the C terminus of the Ca2+ channel α1 subunit (6.Zhang J.F. Ellinor P.T. Aldrich R.W. Tsien R.W. Neuron. 1996; 17: 991-1003Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 7.De Waard M. Liu H. Walker D. Scott V.E. Gurnett C.A. Campbell K.P. Nature. 1997; 385: 446-450Crossref PubMed Scopus (374) Google Scholar, 8.Herlitze S. Hockerman G.H. Scheuer T. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1512-1516Crossref PubMed Scopus (169) Google Scholar, 9.Qin N. Platano D. Olcese R. Stefani E. Birnbaumer L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8866-8871Crossref PubMed Scopus (207) Google Scholar, 10.Zamponi G.W. Bourinet E. Nelson D. Nargeot J. Snutch T.P. Nature. 1997; 385: 442-446Crossref PubMed Scopus (406) Google Scholar, 11.Page K.M. Stephens G.J. Berrow N.S. Dolphin A.C. J. Neurosci. 1997; 17: 1330-1338Crossref PubMed Google Scholar, 12.Furukawa T. Miura R. Mori Y. Strobeck M. Suzuki K. Ogihara Y. Asano T. Morishita R. Hashii M. Higashida H. Yoshii M. Nukada T. J. Biol. Chem. 1998; 273: 17595-17603Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). We first analyzed the interaction of both proteins (Ca2+ channel β and G protein β subunit) on the α1-loopI–II of the P/Q-type channel with the MTH system (Fig. 1h). P/Q-type channel loopI–II and Gβ2 (0.24 ± 0.04 (n = 35)) induced a YFP fluorescent signal, which was significantly weaker than the signal induced with β1b (0.45 ± 0.03 (n = 53)). In contrast, no YFP fluorescent signal was detected for coexpression of L-type channel α11.2-loopI–II and G protein β2 subunits. This result was expected, because G protein βγ subunits do not interact with the L-type channel α1 subunits. To verify the results observed with the MTH system, we compared and analyzed the direct interaction of the α1-loop-I–II with the Ca2+ channel β and G protein β subunits using the three cube FRET method between CFP-tagged donor proteins (loopI–II) and YFP-tagged acceptor proteins (β1b/Gβ2) (Fig. 2). We calculated independently two FRET-based values according to a modified version of Erickson et al. (32.Erickson M.G. Alseikhan B.A. Peterson B.Z. Yue D.T. Neuron. 2001; 31: 973-985Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). First, we determined the average FRET value (Fig. 2b); second, we determined the maximal FRET (FRETmax) value for the interacting protein pairs (Fig. 2, c + d) to qualitatively compare the protein interaction with other interactions examined. Differences in the FRETmax values correspond to a difference in the affinity of the interaction or the distance and/or orientation between the donor relative to the acceptor protein. As shown in Fig. 2b, the interaction between P/Q-type channel α12.1-loopI–II with β1b subunits (FRET 3.45 ± 0.11 (n = 328)) induced a significantly stronger FRET signal than the interaction between α12.1-loopI–II with the Gβ2 subunit (FRET 2.49 ± 0.08 (n = 335)). In contrast, cotransfection of L-type channel α11.2-loopI–II-CFP and Gβ2-YFP did not result in an average FRET signal larger than 1, indicating that these two fusion proteins do not interact (Fig. 2b). Furthermore, cotransfection of CFP and YFP and cotransfection of the donor constructs with YFP or the acceptor constructs with CFP also did not result in a FRET signal larger than 1 (see the Supplemental Material), indicating that the FRET signals measured for the interacting proteins are due to the interaction between donor and a
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